NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite-stage Plasmodium falciparum parasites isolated by saponin-permeabilisation of the host erythrocyte. One-dimensional (1)H NMR spectroscopy and four two-dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the (1)H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological alpha-amino acids; 4-aminobutyric acid; mono-, di- and tri-carboxylic acids; nucleotides; polyamines; myo-inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media.